This invention relates to providing a microchannel chip device which will be able to perform a large number of bio molecule tests simultaneously, as well as producing a uniform test environment for each biomolecule test and eliminate the statistical test to test variations.
It is known in fluid dynamics that, due to the viscosity of the biological sample containing fluid, which is usually water, the dynamic pressure to pass this fluid through and into the multiple channel glass panel increases as the microchannel diameter is reduced and the glass plate thickness increases. Threshold values are such that, below 10xcexc in channel diameter, increase in vacuum pressure is required to force water through the microchannels, and also structural integrity of the glass sample then becomes problematic. However, on the other side, by increasing channel diameter beyond 10xcexc and reducing the thickness of the glass plate, vacuum pressure is still required but to a lesser extent, while undesirable artifacts are generated in particular increased diffuse halos around the top access mouth of the channels. These undesirable artifact halos considerably deteriorate both the image quality and the sensitivity of the test.
It is noted that fluid dynamics in a microchannel are not the same as those in diametrally larger tubes, e.g. a water filled coffee mug. Indeed, because of the larger inner diameter of a coffee mug, when a water filled coffee mug is tilted from an upright condition to a laterally inclined position, the top surface menisk of the volume of water will not concurrently tilt and thus will remain parallel to the ground in both instances, although the longitudinal axis of the mug is no longer vertical in its tilted condition. On the other end, due to surface tension properties and viscosity of the water and due to the micrometer grade diameter of the microscopic (micro-) channel, when a microchannel is tilted from an upright condition to a laterally inclined condition, the menisk will not stay parallel to the ground as it did in larger diameter cylinder such as a coffee mug, but will tilt with the tilted microchannel so that the perpendicular axis to the top surface menisk of the water volume inside the tilted microchannel will remain coaxial to the longitudinal axis of the tilted microchannel.
Existing devices for binding a target molecule comprise a substrate having a multiplicity of discrete tubes extending transversely therethrough. These tubes extend orthogonally to the top surface of the substrate. A first binding reagent is immobilized on the walls of a first group of tubes, while a second binding reagent is immobilized on the walls of a second group of the tubes. Such device is for use in the identification or characterization of nucleic acid sequences through nucleic acid probe hybridization with samples containing an uncharacterized polynucleic acid, e.g. recombinant DNA, polymerase chain reaction fragments, etc . . . as well as other biomolecules.
In these known tubes, their diameter ranges between about 0.03 to 10xcexc. The reason for the top threshold diameter value is that if your have upright tubes or channels, any diameter larger than about 10xcexc will enlarge optical halo artifacts at the top access mouth of the tubes, and accordingly will bring, much reduced sensitivity.
During the 1990s, microfabrication technology has enabled miniaturization and automation of manufacturing processes in numerous industries. The impact of microfabrication technology in biomedical research can be seen in the growing presence of microprocessor controlled analytical instrumentation and robotics in the laboratory engaged in high throughput genome mapping and sequencing (see the current xe2x80x9cHuman Genome Projectxe2x80x9d, with its first phase just completed). Optical detection of fluorescent labelled receptors is employed inter alia in detection for sequencing. Detection can be achieved through use of a charge coupled device array, or confocal laser imaging technology such as DNA scope (TM).
Capillary tube glass arrays are already in use as high surface area nanoporous support structures to tether DNA targets or probes for hybridization. Such capillary tube glass wafers contain a regular geometric array of parallel holes or tubes as small as 33 nanometers in diameter, or as large as several micrometers in diameter. These holes or tubes serve as sample wells for placement of a substantially homogeneous sample of a biomolecule within each hybridization site. The orifices are fabricated using excimer laser machining.
However, such prior art microscopic detection devices usually require charged coupling devices, and cannot scan the full sample area. This is because, since you have vertical micro-channels, the diameter thereof larger than 10xcexc will produce much larger optical halo artifacts and will bring about much diminished microscopic sensitivity. This is why the claimed microchannel diameter in the Beattie patent is limited to a range from 0.03 to 10xcexc.
Methods are also known in the art for delivering sub-nanoliter microdroplets of fluids to a surface at submicron precision. A microjet system or a microspotter, capable of delivering subnanoliter DNA solution to the wafer surface, can thus be employed.
Moreover, in the field of biotechnology, there is an increasing use of biochips for detection of macromolecules such as DNA and proteins. Amongst the various numbers of biochips the flow-through bio-chip is preferred, because of the advantage in terms of speed of the reaction and sensitivity associated with it. Mostly these biochips are made from cylindroid and cross-sectionally polygonal channels that are extended through the length of the panel chip. The diameter of these channels may vary from a few to several hundred micrometers, but usually about 25xcexc.
Most of the testing solutions such as serum, are not homogeneous in nature and contain impurities as the entire cell or cell particles. Some are very viscous in nature and have difficulties passing through small pores of micrometer size. Serum contains fibrin, which is the long strands of wire mesh adapted to entrap the cells. These fibrin strands may be present in serum even after separation. Other impurities like leukocytes with a diameter of 35xcexc and thickness of a few micrometers, may clog the cylindrical pore. If the pore is in the shape of a slot with a length of e.g. 40xcexc and a width of 3xcexc, it thus has opening area of 120 square xcexc which can easily accommodate the passage of white blood cells (WBC) through the panel chip. Cylindrical pores with a diameter of 40xcexc have opening area of (20xc3x9720xc3x973.141592xcexc) or approximately 1,256xcexc2, being ten times bigger than the slot form.
Indeed, the bigger the diameter of cylindrical channels, the worst image deterioration occurs in scanning. The opening area and dead space may be interchangeable terms. Usually, opening area is the area of open space (or empty space) that extends through the chip from the surface or the surface area which separates these channels. The inner surfaces of these pores are areas in which most binding occurs. A cylindrical pore with diameter of 2xcexc and a constant thickness K has opening area of (1xc3x971xc3x973.141592) or approximately 3 square micrometers with inner surface area of (2xc3x97Kxc3x973.141592) i.e. approximately 6K or 6. Since both panel chips have the same thickness, so the K would remain the same. If one increases the diameter of a cylindrical pore to 200xcexc, the opening area would be (100xc3x97100xc3x973.141592) i.e. approximately 30,000 square micrometers, and inner surface would be (200xc3x973.141592) i.e. approximately 600xcexc2. From these calculations it is understood that 100 folds increase in diameter of cylindrical pore shape from 2 to 200xcexc, has resulted in a 10,000 times increase in opening area (from 3 to 30,000) which is considered to be useless area where no binding occurs, and 100 times for inner surface area (from 6 to 600) where the binding occurs and is considered to be the useful area.
The increase of this opening area or useless area which occupies empty space, will compromise the structural integrity of panel chips and make it more prone to breakage and deterioration of image quality. It also contributes to building of arrays of lesser density, since less if not any empty spaces will remain in the chip. If a pore is in the shape of a slot such as rectangle with the length of 10xcexc and width of 2 microns and the thickness of K, it has opening area of (10xc3x972) i.e. 20 square micrometers, and the inner surface area of (10+10+2+2) i . . . 24K, or 24xcexc2 as K stays constant. If the length of slot increases to 200 from 10xcexc, while keeping the width constant at 2xcexc, the opening area would be (200xc3x972) i.e. 400 square micrometers, and the inner surface area of (200+200+2+2) K i.e. 404xcexc2 (K is the thickness of panel chips and stays the same).
Therefore, 20 folds increase in length of slot from 10 to 200xcexc, in order to accommodate the passage of the larger particle size through the channel such as the cell, and viscous solutions has resulted in 20 folds increase in opening area or useless area where no binding occurs (from 20 to 400xcexc2). At the same time, the inner surface area where the binding occurs and is considered as useful area, has increased approximately 17 times (from 24 to 404xcexc2). This clearly demonstrates the superiority of slot form channels over other classical cylindrical shape pores, wherein in slot channels, the ratio of inner surface of channels (useful area) to open area (useless area) stays the same (17/20) compared to (100/10,000) in cylindrical pores, in which there is a one hundred times increase in useless area. But since there is also more slot channels FIG. 16 (100 and 104) in a given area than one single cylindrical channel, therefore, more inner surface area is available for molecule to bind to. Thus, the more intense the image would be.
Therefore, in pores with cylindrical shapes, this drastic exponential increase in ratio of useless area (where no binding occurs) to useful area where binding occurs in order to accommodate the passage of larger particles and cells through the channels, is detrimental and generates limiting factors in increasing the pore size of cylindrical forms, and also incorporates more empty space into the panel chip. This limiting factor to enlargement of cylindrical pores is a contributing factor in blocking of pores and more assisted vacuum control. Any attempt to ignore this limiting factor and build the larger pores, leads to exponential increase in ratio of empty space to useful area and contributes to image deterioration and undermines the structural integrity of the chips. On the contrary, the slot forming channels do not have this negative impact in size enlargement and are capable of acting as wave-guide to carry the fluorescent excited light from the inner area to the surface, while allowing the passage of larger particles and impurities and more viscous solutions. The slot forming channels provide maximum binding inner surface area per pixel, compared to cylindrical pores, and therefore do not contribute to empty space as much as cylindrical pores where there is more inner surface per pixel, so more binding occurs. Therefore, each pixel shows more intensity in slot channels.
An important object of the present invention is therefore to improve upon the above-noted prior art technology, by providing a device which will be able to perform a large number of bio molecule tests simultaneously, as well as producing a uniform test environment for each biomolecule test and eliminate the statistical test to test variations.
A further important object of the present invention is to use the capillary tube as an environment that can produce an internal reflection known as xe2x80x9cpipping effectxe2x80x9d, so as to increase sensitivity and resolution of biomolecule detection.
Still another object of the invention is to use the capillary tube as an environment in which samples and reagents flow through, to increase the interactions between biomolecules so as to reduce the incubation time and increase the sensitivity and resolution at the same time, to thus enable use of a more diluted sample for the same efficiency.
An object of the invention is to provide a structure suited for making a device for binding macromolecules while allowing the passage of other impurities to prevent clogging of pores and further facilitating flow-through of solution to be tested.
An important object of the present invention is to provide a large variety of different patterns and shapes of channels layout through the thickness of the microchip, in each unit cell, so as to enable matching of channels layout with the shapes of microscopic impurities in testing solutions.
A further important object of the present invention is to provide a better flow for more viscous solutions in microchip channels, by increasing the length of channel slots while keeping the width to a minimum.
Another object of the invention is to provide slot channel shape generating a maximum binding environment within a minimum opening area. This will result in increase in the number of molecules binding per pixel, since more inner surface is available which increases the intensity of pixel and provides better sensitivity.
An important object of the invention is to provide a sharper and more uniform microchip channel imaging. This will improve the dynamic range in greater detail, and provide better distinction between positive and negative spots and leave less to guesswork.
A general object of the invention is to reduce labour costs and required effective sample volume associated with operation of such devices.
According to the invention, there is disclosed a rigid panel chip for supporting biological samples for observation with a microscope, said glass panel defining a top flat surface, a bottom bearing surface, and a plurality of unit cells extending generally parallel to each other from said top to bottom surfaces, each of said unit cells defining a layout at said top surface of at least two channels arranged in plan view generally symmetrically relative to one another, each of said channels defining a top access mouth for ingress of said biological samples and having such an inner diameter as to accommodate flow through viscosity of a biological sample containing fluid;
wherein a sharper and more uniform panel chip channel imaging is achieved.
Preferably, the number of said channels in each of said unit cells can vary between about two and thirty.
The substrate for fabrication of the panel chip may be selected from polymers, plastics, polypropylene, parylene, polyester, polyimide, polyurethane, synthetic resins, polyethylene, polystyrene, glass, silicon dioxide, fused silica, borosilicate, metal, and aluminum.
Said channels layout at said panel top surface in a given unit cell could be selected from:
at least three straight slot channels, extending parallel to one another;
an air fan like layout of arcuately shaped xe2x80x9cbladexe2x80x9d channels, with a central circular xe2x80x9chubxe2x80x9d channel forming the top end of a cylindroid channel;
a number of concentrically disposed arcuate channels, with a central circular channel forming the top end of a cylindroid channel; and
a number of cross-shaped disposed straight channels.
Preferably, said channels layout at said panel top surface in a given unit cell includes a number of star shaped disposed straight channels. A pair of straight channels may be added, being much shorter than said star shaped disposed channels and disposed parallel to one another centrally of said star shaped disposed channels. A plurality of circular channels could be disposed concentrically of said star shaped disposed channels and forming the top end of cylindroid channels. A plurality of additional shorter straight segments channels could each be disposed concentrically of said star shaped disposed channels in between a pair of successive said star shaped disposed channels but at a distance from the center of the unit cell.
Alternately, said channels layout at said panel top surface in a given unit cell could include a number of C-shaped channels successively disposed circumferentially thereof in the same trough-facing circular direction. A number of circular channels could then be disposed within said C-shaped channels and forming the top end of cylindroid channels. A plurality of shorter straight segments channels could also be disposed within said C-shaped channels.
Again alternately, said channels layout at said panel top surface in a given unit cell may include a pair of triplets of C-shaped channels disposed in facing opposing register relative to one another. A plurality of circular channels could be sparsely settled about said C-shape channels and forming the top end of cylindroid channels.
Alternately, a plurality of shorter straight channels could be sparsely settled in between each pair of successive C-shape channels.
Said channels layout at said panel top surface in a given unit cell could rather include a plurality of radially extending waterdroplet like channels.
Said channels layout at said panel top surface in a given unit cell could also include a few waterdroplet like channels, disposed parallel to one another.
In a more general sense, the invention could also be directed to a rigid panel chip for supporting biological samples for observation with a microscope, said glass panel defining a top flat surface, a bottom bearing surface, and a plurality of unit cells extending generally parallel to each other from said top to bottom surfaces, each of said unit cells defining at least one slot channel, said channel defining a top access mouth for ingress of said biological samples and having such an inner diameter as to accommodate flow through viscosity of a biological sample containing fluid; said slot channel having a shape selected from a straight slot, an arcuate slot and a water droplet like slot;
wherein a sharper and more uniform panel chip channel imaging is achieved.